As a result of classical strain improvements, penicillin production has increased enormously over the last four decades. These classical strain improvements were primarily based on random mutagenic treatments of Pencillium chrysogenum and subsequent selection for mutants that produced more penicillin. The development of cloning techniques however has added a potentially powerful new tool to further improve penicillin production in this fungus.
Penicillin is produced by the filamentous fungus P. chrysogenum in several enzymatic steps (e.g. E. Alvarez et al., Antimicrob. Agents Chemother. 31 (1987) pp. 1675-1682). These steps are shown in FIG. 1. Throughout this specification is meant by genes directly involved in the biosynthetic pathway, those genes that encode the enzymes active in the several steps leading to the production of a secondary metabolite, so in case of the production of penicillin G or V, the genes encoding the enzymes shown in FIG. 1 are meant. The first reaction is the formation of the tripeptide .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine from .alpha.-amino adipic acid, cysteine and valine. The enzyme that is responsible for this reaction is the ACV synthetase (hereinafter referred to as ACVS), a large multifunctional enzyme. The tripeptide is cyclised by the action of the isopenicillin N synthetase (hereinafter referred to as IPNS) or cyclase. The reaction product is isopenicillin N, a compound that contains the typical .beta.-lactam ring structure and that possesses antibacterial activity. The final step in the formation of penicillin is the exchange of the .alpha.-aminoadipic acid side chain of isopenicillin N by a hydrophobic side chain. The hydrophobic side chains commonly used in industrial production are phenylacetic acid, yielding penicillin G and phenoxyacetic acid, yielding penicillin V. The side chain exchange has been proposed to be a reaction catalysed by a single enzyme (A. L. Demain (1983) in: A. L. Demain and N. A. Solomon (ed), Antibiotics containing the .beta.-lactam structure I. Springer Verlag, Berlin; pp. 189-228). However, a two step reaction involving 6-APA as an intermediate is also possible (E. Alvarez et al., vide supra). The enzyme that has been identified to be involved in the final reaction is the acylCoA: 6-APA acyltransferase (hereinafter referred to as AT); this enzyme has been purified to homogeneity (E. Alvarez et al., vide supra). The involvement of a second enzyme, catalysing the reaction from IPN to 6-APA, cannot yet be confirmed nor excluded.
It is not clear either whether one or more enzymatic reactions are rate limiting in the process of penicillin biosynthesis, and if so, which enzymatic steps are involved.
Since the penicillin biosynthetic route begins with three amino acids, which each in their turn are part of other metabolic routes, regulatory steps in these routes will also influence the biosynthesis of penicillin. On the other hand, the production of penicillin is subject to a complex mechanism of carbon catabolite repression and nitrogen source control (J. F. Martin et al. In: H. Kleinkauf, H. von Dohren, H. Donnauer and G. Nesemann (eds), Regulation of secondary metabolite formation. VCH Verlaggesellschaft, Weinheim (1985), pp. 41-75). Regulatory proteins may also be involved in these types of regulation. These regulatory proteins and the proteins regulated by them are defined to be indirectly involved in the biosynthetic pathway of a secondary metabolite, in this case penicillin.
Until recently, the gene of only one of the enzymes active in the biosynthetic pathway to penicillin G, the isopenicillin N synthetase (IPNS) or cyclase, had been cloned and sequenced (L. G. Carr et al., Gene 48 (1986) pp. 257-266), using the corresponding Acremonium chrysogenum gene (S. M. Samson et al. Nature 318 (1985) pp. 191-194). The latter gene was cloned and identified by purifying the IPNS protein, determining the amino-terminal amino acid sequence, preparing a set of synthetic oligodeoxyribonucleotides according to this sequence and probing a cosmid genomic library with these mixed oligodeoxyribonucleotides (S. M. Samson, vide supra).
The isolated genes encoding IPNS from both Penicillium chrysogenum and Acremonium chrysogenum have been used for strain improvement. In Penicillium chrysogenum an enhanced enzyme activity has been demonstrated; however no stimulation of penicillin biosynthesis has been found (P. L. Skatrud et al, Poster presentation 1987 annual meeting of the Society of Industrial Microbiology, Baltimore, August 1987, Abstract published in SIM News 37 (1987) pp. 77). In Acremonium chrysogenum similar results have been obtained (J. L. Chapman et al, (1987), in: Developments in Industrial Microbiology, Vol. 27, G. Pierce (ed), Society of Industrial Microbiology; S. W. Queener, 4th ASM conference on the Genetics and Molecular Biology of Industrial Microorganisms, Bloomington, October 1988, Proceedings will appear in 1989).
Therefore, up to now no evidence has been obtained that the IPNS gene can be used to obtain increased production of penicillin or cephalosporin by gene amplification.
It has been documented that the biosynthesis of .beta.-lactam antibiotics is subject to glucose repression (J. F. Martin and P. Liras, TIBS 3 (1985), pp. 39-44). This repression by glucose has been unequivocally established for the formation of the tripeptide by the ACVS and for the activity of the IPNS (Revilla et al., J. Bact. 168 (1986), pp. 947-952). For acyltransferase, on the other hand, the data are less convincing. Revilla et al (vide supra) report that AT is not subjected to glucose repression, but other data suggest that AT activity is absent, or at least decreased, in the presence of glucose (B. Spencer and T. Maung, Proc. Biochem. Soc. 1970, pp. 29-30).
It is unknown at which stage of the expression the repression by glucose is exerted; this can be at the transcriptional or at the translational level. If the former regulation applies, differences in mRNA levels between producing and non-producing cultures could be employed to isolate genes, involved in the biosynthesis of penicillin. This method for the isolation of genes involved in the biosynthesis of secondary metabolites is the subject of U.S. Pat. No. 5,108,918, issued Apr. 28, 1992, entitled: "A method for identifying and using biosynthetic or regulatory genes for enhanced production of secondary metabilites" and which is incorporated here by reference.
Clustering of antibiotic biosynthetic genes has been described for Streptomycetes. Some examples are the clustering of the genes involved in the biosynthesis of actinorhodin by S. coelicolor (F. Malpartida and D. A. Hopwood, 1984, Nature 309, 462-464) or in the biosynthesis of tetracenomycin C by S. glaucescens (H. Motamedi and C. R. Hutchinson, 1987, Proc. Natl. Acad. Sci. U.S.A. 84, 4445-4449).
In fungi, the gene organization of .beta.-lactam biosynthetic genes has been investigated by genetic analysis of mutants, impaired in penicillin biosynthesis. In Aspergillus nidulans, four loci have been identified that are involved in penicillin biosynthesis (npe A, B, C and D); these loci have been positioned on four different linkage groups (i.e. chromosomes), viZ. VI, IV, III and II, respectively (J. F. Makins et al., 1980, Advances in Biotechnology 3, 51-60; J. F. Makins et al, 1983, Journal of General Microbiology 129, 3027-3033). In Penicillium chrysogenum five loci have been identified (npe V, W, X, Y and Z), these loci have been positioned on three linkage groups, viz. I (npe W, Y, Z) and two others containing npe V and npe X, respectively (P. J. M. Normansell et al, 1979, Journal of General Microbiol. 112, 113-126; J. F. Makins et al, 1980, vide supra). The mutations affecting the ringclosure enzyme (IPNS or cyclase; npe W) and the side chain exchange enzyme (acyltransferase, npe V) are reported to be in separate linkage groups. Hence, the genetic data predict that at least some penicillin biosynthetic genes are spread over the fungal genomes, and clustering of e.g. the cyclase and acyltransferase genes is definitely not anticipated based on these data.